Sarcopenia, a condition linked to aging, involves a gradual and widespread decline in muscle mass and strength or function (Dent et al., 2018). An aging global population poses a major public health challenge, resulting in a rise in age-related illnesses, a decline in life quality, and considerable economic strain from escalating healthcare expenses and the demand for long-term care. Therefore, the prevention and treatment of sarcopenia are very important public health issues, and much attention should be paid to (Cruz-Jentoft and Sayer, 2019).

Current preventive and curative measures for sarcopenia include targeted exercise therapy (Pahor et al., 2014; Bann et al., 2016), adequate nutritional interventions (Tieland et al., 2012; Cermak et al., 2013), and pharmacological treatments. Exercise, in particular, is widely recognized as one of the most effective interventions for sarcopenia, as it helps maintain muscle mass, strength, and function through resistance and aerobic training. The development of sarcopenia has long been a focus of scientific study due to the incomplete understanding of the molecular processes involved, and the current absence of successful treatments for preventing or treating sarcopenia. The pathophysiological mechanisms of sarcopenia in the elderly are complex. These mechanisms include oxidative stress, mitochondrial dysfunction, imbalance of protein metabolism, inflammation, apoptosis, satellite cell dysfunction, decreased motility, diminished neuromuscular function, and aging-related humoral changes (Li et al., 2018). The complex pathophysiological mechanisms that can occur during human aging provide a wide range of potential therapeutic targets for sarcopenia treatment (Cruz-Jentoft and Sayer, 2019); however, the discovery of multiple targets and signaling pathways in the pathogenesis of sarcopenia has also complicated the clinical transformation. Hence, it is of utmost importance to recognize the junction of these processes.

Sarcopenia is an involuntary loss of skeletal muscle mass and strength associated with aging (Shaw et al., 2017). This condition results in a decline in bodily functions and reduces the stability and stress capacity of the internal environment, leading to senescence. ER stress plays a significant role in the aging process, not only through the accumulation of damaged proteins (Matos et al., 2015) but also via other mechanisms, such as the involvement of the inner nuclear membrane protein SUN2, which activates ER stress in premature aging (Vidak et al., 2023). Additionally, aging-related mitochondrial dysfunction and sphingolipid imbalances have been shown to exacerbate ER stress, further contributing to cellular decline (Chen et al., 2023). Ikeyama et al. (Ikeyama et al., 2003) reported an increased basal expression of C/EBP homologous protein (CHOP) in the liver and pancreas of mice during senescence. These results suggest that chronic ERS is common in senile bodies.

CHOP is a well-characterized transcription factor upregulated during severe and sustained ER stress, primarily through the PERK-eIF2α-ATF4 pathway (Ikebe et al., 2020). Elevated CHOP expression promotes apoptosis by suppressing anti-apoptotic proteins (e.g., Bcl-2), inducing pro-apoptotic factors (e.g., Bim), and increasing oxidative stress through reactive oxygen species production (Ghosh et al., 2012; Noh et al., 2012). Thus, CHOP plays a pivotal role in the cellular injury associated with prolonged ER stress, including skeletal muscle pathology.

In eukaryotic cells, the endoplasmic reticulum plays a vital role as a membrane organelle in the processes of protein synthesis, folding, and secretion. Either endogenous or exogenous stimuli leads to dysfunction of the endoplasmic reticulum in folding proteins. Numerous improperly folded proteins build up in the endoplasmic reticulum, causing a disruption in its balance, known as ERS (Lam et al., 2020). The activation of the unfolded protein response (UPR) controls the subsequent pathway through three primary pathways: protein kinase RNA-like ER kinase (PERK)-eIF2α, inositol-requiring enzyme 1α (IRE1α)-x-box-binding protein 1 (XBP-1), and activating transcription factor 6 (ATF6) (Kato and Nishitoh, 2015). In turn, it regulates apoptosis, inflammation, autophagy, oxidative stress, etc., and helps stressed cells restore homeostasis (Chen and Cubillos-Ruiz, 2021). In the PERK-eIF2α pathway, ATF4 functions as a critical downstream effector. Upon eIF2α phosphorylation, the translation of ATF4 is selectively upregulated, promoting the expression of genes involved in apoptosis, autophagy, and oxidative stress response (Rzymski et al., 2009; Hart et al., 2012). This highlights ATF4’s central role in restoring cellular homeostasis under ER stress conditions.

Salubrinal is a synthetic and highly selective small molecule inhibitor of eIF2α dephosphorylation (480 Da, C21H17Cl3N4OS), which can activate intracellular eIF2α phosphorylation and thus reduce ERS-induced apoptosis. Studies have demonstrated that salubrinal has therapeutic effects on tumors (Kardos et al., 2020; Alsterda et al., 2021), arthritis (Wang et al., 2021), paraquat poisoning (Wang et al., 2018), and neuroprotective effects in cardiopulmonary resuscitation models and cerebral hemorrhage models (Masuri et al., 2020; Zhang et al., 2020). Although studies have confirmed that salubrinal can alleviate many diseases by modulating the eIF2α-related signaling pathway, its role in senile sarcopenia is unclear.

Although it is known that ERS plays a significant role in muscle wasting, precisely regulating ERS to protect muscle cells from damage, remains an unresolved issue. Furthermore, the specific role of the eIF2α/ATF4 signaling pathway, which is part of the ERS response, in muscle wasting is not yet clear. Thus, exploring potential drugs that can effectively modulate this pathway to alleviate muscle cell damage is crucial for developing new therapeutic strategies.

This study aims to explore how salubrinal can protect muscle cells from injury caused by H₂O₂ and to investigate if this protection is linked to the eIF2α/ATF4 signaling pathway. This study aims to uncover the molecular mechanisms through which salubrinal reduces muscle cell damage by examining its effects on MHC1, p-eIF2α, ATF4 expression, and cell apoptosis. We anticipate that salubrinal will effectively inhibit H₂O₂-induced muscle cell injury, reduce cell apoptosis, and exert its protective effects by modulating the eIF2α/ATF4 signaling pathway. This finding will not only offer a new perspective on the molecular mechanisms of muscle wasting but also provide scientific evidence for the development of novel therapeutic drugs targeting muscle wasting.

Previous studies have shown a significant increase in Atrogin-1 and MuRF-1 levels in the gastrocnemius muscle of aged mice compared to younger groups, indicating a strong correlation with muscle atrophy during aging. (Supplementary Figure S3). In this study, we examined the expression of muscle ERS-related proteins GRP78 and CHOP, from the samples of our preveous study, and presents pivotal findings on the protective effects of salubrinal against H₂O₂-induced muscle wasting in cell experiments. Firstly, significant muscle atrophy and ERS were found in the gastrocnemius muscle of aged mice. Secondly, it was found that salubrinal significantly ameliorates H₂O₂-induced damage in myotubes. The study is significant in showing that salubrinal’s protection is linked to the eIF2α/ATF4 signaling pathway, supported by the rise in p-eIF2α and ATF4 levels when salubrinal is present. Additionally, salubrinal was found to reduce muscle cell apoptosis, further underscoring its therapeutic potential in sarcopenia and muscle wasting conditions.

Muscular fibrosis is known to be induced by advanced age. The increase in Atrogin-1 and MuRF-1 expression in aged mice highlights the role of ubiquitin-proteasome pathways in muscle atrophy, consistent with findings from Bodine et al. (2001) and Schiaffino et al. (2013), who emphasized the importance of these pathways in muscle protein degradation. The elevated levels of GRP78 and CHOP suggest an enhanced ERS response in aged muscle, aligning with the work of Chalil et al. (2015) and Deldicque (2013), which pointed out the critical role of ERS in muscle aging and atrophy.

It has been shown that ERS-activated UPR protects stressed cells by reducing protein synthesis and thus mitigating ERS, on the one hand, restoring endoplasmic reticulum homeostasis by decreasing the burden on the endoplasmic reticulum through protein folding to a certain extent; on the other hand, since the former regulatory capacity is limited, it initiates apoptosis under conditions of sustained stress and unrepairable cellular damage to eliminate these stressed cells. UPR is mainly mediated by three endoplasmic reticulum transmembrane receptors: IRE1, PERK and ATF6 (Ramirez et al., 2019). They are transmembrane proteins located in the endoplasmic reticulum membrane. Under normal conditions, they are bound to the chaperone molecule GRP78 and remain in an inactive state. When ERS occurs, unfolded or misfolded proteins recruit GRP78, which results in the dissociation of GRP78 from IRE1, ATF6, and PERK, and after dissociation IRE1, ATF6, and PERK are activated and further activate downstream UPR signaling (Frakes and Dillin, 2017).

Studies have shown that the three main signaling pathways of the UPR (PERK-eIF2α, IRE1α-XBP-1 and ATF6 (Kato and Nishitoh, 2015) tend to be activated in most stressful situations, thus making it more difficult to study the role of the UPR in pathophysiological processes related to skeletal muscle (Pierre et al., 2014). Several recent studies have focused on the PERK-eIF2α signaling pathway. Studies have shown that mice overexpressing PERK, compared to wild-type mice, lose weight and have decreased muscle strength and muscle mass in adulthood, and that sustained activation of the PERK pathway inhibits protein synthesis by promoting phosphorylation of eIF2α leading to a decrease in muscle mass, but enhances expression of antioxidant genes through specific promotion of ATF4 expression leading to healthier skeletal muscle (Miyake et al., 2016). However, mice with specific knockout of PERK also showed decreased muscle mass and muscle atrophy, although knockout of PERK caused a decrease in the ratio of p-eIF2α/eIF2α and an increase in protein synthesis, it also induced the activation of the ubiquitin proteasome pathway, autophagy pathway, and ultimately caused skeletal muscle atrophy (Gallot et al., 2019). Given these seemingly contradictory findings, the role of the PERK-eIF2α pathway in muscle needs to be explored in further studies.

Research has demonstrated that when skeletal muscle cells are exposed to H₂O₂, it leads to harm at the cellular level, such as apoptosis (Yin et al., 2015), oxidative stress (Kang et al., 2015), endoplasmic reticulum stress (Pierre et al., 2014), autophagy (Kaur et al., 2019), and mitochondrial dysfunction (Fan et al., 2010). Our study aimed to target endoplasmic reticulum stress to explore the molecular mechanisms of Sarcopenia, so we chose H₂O₂ stimulation of C2C12 myotubular cells as a model of muscle injury. Our study showed that H₂O₂ could induce myotube injury in a concentration- and time-dependent manner.

Salubrinal, a synthetic endoplasmic reticulum stress inhibitor, is a small molecule compound targeting the eIF2α pathway that selectively binds to the PP1/GADD34 complex, inhibits PP1 activity, preventing the dephosphorylation of eIF2α, and helping to increase the phosphorylation of eIF2α, ultimately reducing apoptosis caused by endoplasmic reticulum stress. Meanwhile, salubrinal is characterized by high safety and selectivity; low cytotoxicity, without toxic cellular effects at a maximum concentration of 100 μmol/L; and as a selective protein phosphatase 1 (PP1)/growth arrest and DNA damage-inducible gene 34 (GADD34) inhibitor, it can only selectively inhibit only a few PP1 complexes in the body and has no effect on dozens of other PP1 complexes (Boyce et al., 2005). Further studies have shown that salubrinal has therapeutic effects on tumors (Kardos et al., 2020; Alsterda et al., 2021), arthritis (Wang et al., 2021), and paraquat poisoning (Wang et al., 2018), but very few studies have been reported on its application in skeletal muscle cytoprotection. In our study, we found that salubrinal’s effect on inhibiting the H₂O₂-induced decrease in MHC1 expression and reducing apoptosis through concentration-dependent mechanisms sheds light on its potential as a therapeutic agent. This finding is in harmony with the research by Matsuoka and Komoike (2015), who demonstrated salubrinal’s efficacy in protecting cells from ERS-induced apoptosis.

Several studies have demonstrated that salubrinal effectively impacts oxidative stress. For instance, salubrinal reduces cell apoptosis caused by oxidative stress by inhibiting eIF2α dephosphorylation, which promotes cell survival (Boyce et al., 2005). This is especially relevant in models of oxidative stress, like H₂O₂-induced cell damage, where reactive oxygen species disrupt cellular homeostasis. Salubrinal maintains higher levels of phosphorylated eIF2α, helping to reduce oxidative burden and preventing protein misfolding under stress. Our study found that salubrinal alleviated the negative effects of H₂O₂-induced oxidative stress in muscle cells, consistent with its protective effects against ROS-induced damage in other cell types (Goswami et al., 2016; Chen et al., 2021). Therefore, it is evident that salubrinal not only protects against endoplasmic reticulum stress but also plays a role in mitigating oxidative stress through its action on the eIF2α/ATF4 signaling pathway.

The involvement of the eIF2α/ATF4 pathway in salubrinal’s protective effects against muscle wasting adds a new understanding of our knowledge in muscle atrophy’s molecular mechanism. It has been shown that phosphorylated eIF2α rapidly and efficiently inhibits translation initiation, thereby effectively blocking most protein synthesis (Harding et al., 1999); the result is a reduction in the entry of newly synthesized proteins into the endoplasmic reticulum, which in turn reduces overloading of the need for folding of newly synthesized proteins. However, upon eIF2α phosphorylation, not all mRNA translation is terminated, and the translation of the transcription factor ATF4 is selectively upregulated; the specific mechanism is that the open reading frame upstream of its mRNA 5′-untranslated sequences, which is formed in a spatial conformation under non-stressful conditions prevents the translation of ATF4’s open reading frame, and the phosphorylation of eIF2α causes the ribosome to phosphorylation of eIF2α causes the ribosome to ignore the initiation of its upstream open reading frame during scanning and directly start the translation of the ATF4 open reading frame. ATF4 has both pro-apoptotic and pro-survival roles, and induces the expression of proteins related to cellular metabolism, redox homeostasis, apoptosis, cellular autophagy, etc., which depend on the extent of ERS and the intracellular environment (Blais et al., 2004). ATF4 regulates methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate dehydrogenase (PHGDH), and phosphoserine aminotransferase 1 (PSAT1) series at the transcriptional level, which are involved in regulating the expression of enzymes related to nicotinamide adenine dinucleotide phosphate (NADPH) production and play an antioxidant role, thus affecting the endoplasmic reticulum homeostasis and cell apoptosis (Wang et al., 2022). ATF4 can also regulate CHOP at the transcriptional level, which is a pro-apoptotic transcription factor that regulates the transcription of apoptosis-related genes and whose sustained activation induces apoptosis (Ikebe et al., 2020).

ATF4 is involved in the endoplasmic reticulum stress response, protein folding, and calcium signaling. Several studies indicate that ATF4 regulates calcium homeostasis and pathways related to cellular survival and stress responses. For example, Xiang et al. reported that the eIF2α-ATF4 pathway is activated by changes in the calcium environment and is involved in bone regeneration, indicating a close relationship between calcium signaling and this pathway (Xiang et al., 2021). Moreover, Hinton et al. demonstrated that ATF4-dependent increases in mitochondrial-endoplasmic reticulum tethering after OPA1 deletion highlight ATF4’s potential role in calcium regulation (Hinton et al., 2024). Although our study did not directly examine the effects of H₂O₂ + Salubrinal treatment on calcium signaling, these findings suggest that salubrinal may regulate calcium signaling by enhancing p-eIF2α and ATF4 expression. This aspect warrants further investigation in future studies.

Our research revealed that salubrinal improved H₂O₂-induced muscle damage and enhanced ATF4 expression in muscle cells. Additionally, the levels of CHOP protein expression showed a slight decrease without reaching statistical significance. This indicates that salubrinal’s muscle protection benefits may be attributed to the promotion of survival-related genes through ATF4.

Besides its involvement in the eIF2α/ATF4 signaling pathway, ATF4 also regulates mitochondrial function, shape, and content in skeletal muscle cells. Recent studies, including Memme et al. (2023), show that ATF4 affects mitochondrial dynamics during muscle cell differentiation, which directly influences mitochondrial content and shape. Additionally, ATF4 is crucial in the mitochondrial stress response, as noted by Quirós et al. (2017), where it modulates different mitochondrial proteins in response to cellular stress. Furthermore, Hinton et al. (2024) found that ATF4-dependent tethering between mitochondria and the endoplasmic reticulum is vital for maintaining cellular homeostasis during stress. These findings indicate that salubrinal’s protective effects against oxidative stress and muscle cell apoptosis may involve ATF4-mediated regulation of mitochondrial function.

Our study observed an increase in CHOP expression following H₂O₂-induced stress, consistent with previous findings linking elevated CHOP levels to muscle atrophy and cell death (Zheng et al., 2023; Michel et al., 2025). However, the reduction in CHOP expression following salubrinal treatment did not reach statistical significance, indicating a more complex regulation of CHOP under our experimental conditions. Our results indicated the necessity for future studies to thoroughly investigate CHOP’s regulatory network and its precise role in muscle injury and recovery.

ATF4 exerts dual roles in skeletal muscle, mediating either adaptive responses or apoptosis depending on stress intensity and duration. In our study, ATF4 expression correlated with protective effects of salubrinal, suggesting a context where ATF4 activation favors cell survival. Previous research has demonstrated that salubrinal’s modulation of ATF4 can ameliorate degenerative changes in lung and neurons (Wang et al., 2018; Mizugaki et al., 2022), supporting its potential translational value.

Overall, our tests showed that salubrinal, a compound that blocks endoplasmic reticulum stress, reduces damage to myotube cells caused by H₂O₂. This effect is likely due to how it affects the eIF2α/ATF4 pathway, leading to anti-apoptotic outcomes, as illustrated in Figure 9. Our initial studies investigated the possible use of salubrinal as a treatment for Sarcopenia.

Although the study shows strong support for salubrinal’s protective effects, several limitations need to be addressed in future research. We acknowledge the limitation of our study in not directly examining the roles of additional markers such as ATF6, XBP1, pPERK, p62, and LC3B, which could provide deeper insights into autophagy and unfolded protein response mechanisms. Future research should focus on these additional pathways to fully elucidate the complex interaction between ER stress, oxidative stress, and autophagy in muscle wasting. Furthermore, the downstream effects of activating the eIF2α/ATF4 pathway, particularly the roles of CHOP and other stress-responsive genes, require further investigation to fully elucidate salubrinal’s mechanism of action. Future studies should also investigate the long-term effects of salubrinal treatment on muscle function and atrophy in aging and disease contexts.

Despite supporting evidence from previous studies (Lee et al., 2021; Mizugaki et al., 2022) for the use of 300 µM H₂O₂ for oxidative stress induction in muscle cells, we recognize the lack of ROS or viability assay data as a limitation in our model validation. Future studies will include ROS quantification and cell viability testing to provide direct evidence of oxidative injury.

While we included TUNEL staining to evaluate apoptosis, we acknowledge that assessing specific injury markers and oxidative stress indicators would provide deeper insights into the mechanisms of H₂O₂-induced muscle injury. Previous studies have confirmed the elevation of these markers in H₂O₂-induced muscle cell injury models, supporting the role of oxidative stress in mediating cellular damage (Pierre et al., 2014; Kang et al., 2015; Yin et al., 2015). Due to resource limitations, we were unable to perform these assays; however, we consider this an important limitation. In future studies, we will incorporate these indicators to more precisely characterize the cellular injury mechanisms and better evaluate the protective effects of salubrinal in oxidative stress-related myopathy.

Although we observed concurrent activation of ER stress and oxidative stress following H₂O₂ treatment, we did not include experiments involving antioxidant intervention to delineate their causal relationship. Prior research has shown that mitigating oxidative stress can suppress ER stress pathways in multiple cell types (Wang et al., 2016; Esmaeili et al., 2022). In future studies, we aim to investigate whether pharmacological inhibition of oxidative stress using agents such as N-acetylcysteine or mitoTEMPO can modulate ER stress and apoptosis in our model. Such work would further clarify the mechanistic role of salubrinal in the context of oxidative and ER stress cross-talk.

Salubrinal-treated cells showed no overt signs of cytotoxicity, consistent with previous reports suggesting its relative safety at the concentration used (Boyce et al., 2005; Matsuoka and Komoike, 2015). However, further dose-response studies will be conducted to confirm its safety profile.

While our study demonstrates the regulatory effects of salubrinal on the eIF2α/ATF4 signaling pathway in vitro, we acknowledge the absence of in vivo validation as a limitation. Previous research has confirmed the relevance of this pathway in animal models of muscle atrophy (Adams et al., 2017; Montori-Grau et al., 2022; Zheng et al., 2023). In future work, we aim to establish in vivo models to further verify the protective role of salubrinal in skeletal muscle and enhance the translational value of our findings.

In conclusion, we found an increase in gastrocnemius ERS levels in elderly mice, confirming that salubrinal can improve H₂O₂-induced myotube cell injury, and the mechanism may be related to salubrinal’s regulation of the eIF2α/ATF4 pathway. Further study of salubrinal’s muscle-protecting role in animals is needed in the future.